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Review Article
Transfer RNA as a Source of Small Functional RNA
Megumi Shigematsu, Shozo Honda and Yohei
Kirino*
Computational Medicine Center, Department of
Biochemistry and Molecular Biology, Sidney Kimmel
Medical College, Thomas Jefferson University, USA
*Corresponding author: Yohei Kirino, Computational
Medicine Center, Department of Biochemistry and
Molecular Biology, Sidney Kimmel Medical College,
Thomas Jefferson University, USA
Received: September 09, 2014; Accepted: October 04,
2014; Published: October 08, 2014
Abstract
Since their discovery in the 1950s, transfer RNAs ( tRNAs ) have been
best known as adapter molecules that play a central role in translating genetic
information. However, recent biochemical and bioinformatics evidence has led
to a previously unexpected conceptual consensus that tRNAs are not always
end products; they further serve as a source of small functional RNAs. In many
organisms, specific tRNA fragments are produced from mature tRNAs or their
precursor transcripts not as random degradation products, but as functional
molecules involved in many biological processes beyond translation. In this
review, we summarize recent studies of tRNA fragments that have provided
new insights into tRNA biology by examining the molecular functions of tRNA
fragments and proteins with which they interact.
Keywords: tRNA; tRNA fragment; tRNA half; tRF; Argonaute
Introduction
The groundbreaking development of next-generation sequencing
(NGS) technologies has dramatically advanced our understanding of
the cellular transcriptome, revealing that non-protein-coding regions
of the genome are widely transcribed, and the generated non-coding
RNAs (ncRNAs) play important roles in normal biological processes
and diseases [1]. Within the diverse group of ncRNAs, the functional
significance is particularly evident for small regulatory RNAs which
direct the highly-specific regulation of gene expression by recognizing
their complementary RNA targets [2-4]. To date, the following three
major classes of small regulatory RNAs have been reported: micro
RNAs (miRNAs), short-interfering RNAs (siRNAs), and PIWIinteracting RNAs (piRNAs). The definitive features of these RNAs
are their short lengths of 20–31 nucleotides (nt) and their interaction
with Argonaute family proteins to form effectors ribonucleoprotein
complexes. miRNAs, the best-studied class of small regulatory
RNAs, repress complementary target mRNA expression, which is
estimated to regulate the expression of most protein-coding genes
[5, 6]. Thus, small regulatory RNAs constitute one of the most
abundant gene expression regulators and exhibit a tremendous
impact on all biological processes by shaping the transcriptome.
Although such small RNAs have been the focus of much attention
over the recent years, NGS studies combined with RNA biochemical
studies have revealed the existence of many different other functional
small ncRNAs in the cellular transcriptom'e, including small RNA
fragments from transfer RNAs (tRNAs), which we will highlight in
this review.
L-shaped tertiary structure. Over 500 tRNA genes are encoded in the
human genome [8], and tRNAs have long half-lives, estimated on
the order of days in tissues[9, 10]. Active transcription from multiple
sites and high stability place tRNAs among the most abundant RNA
molecules, occupying around 15% of the cellular RNA transcriptome.
Considering their abundance and well-defined biological role
in translation, it is not surprising that RNA fragments from tRNAs
were regarded as non-functional degradation intermediates for a long
time. The apparent presence of abundant tRNA fragments in early
NGS studies was often ignored. However, the combined biochemical
evidence from many years of tRNA biology has recently brought the
field to a previously unexpected conceptual consensus: specific tRNA
fragments are widely expressed not as random degradation products
but as functional molecules in many different cells of various
organisms [11-15]. The expression of tRNA fragments does not
usually affect mature tRNA pools. Instead, it is involved in normal
biological processes beyond translation and in diseases; thus, studies
of tRNA fragment have provided new insights into tRNA biology.
The first evidence of the presence of functional tRNA fragments
was reported in 1999; conditioned medium from human urinary
bladder carcinoma cells contained tRNA fragments that exhibited an
inhibitory effect on endothelial cell growth [16]. In this review, we
will summarize the subsequent accumulation of findings, as displayed
in Table 1, concerning functional tRNA fragments whose molecular
functions and/or associated proteins have been experimentally
examined and validated.
Unexpected Expansion of the tRNA World
Classification of tRNA Fragments: tRNA
Halves and tRFs
tRNAs are universally expressed in all three domains of life,
and play a central role in gene expression as adapter molecules 'that
translate codons in mRNA into amino acids in protein. Since their
discovery in the 1950s, extensive studies have clearly defined their
biological properties [7]. tRNAs are 70–90 nt in length and form
as cloverleaf secondary structure containing three major loops (D-,
T-, and anticodon loops) and four stems (acceptor-, D-, T-, and
anticodon stems) (Figure 1). These loops and stems fold into an
Mature tRNAs are produced from precursor tRNAs (pre-tRNAs),
which undergo several steps during maturation [11]. In the first step,
pre-tRNAs are transcribed from tRNA genes by RNA polymerase
III. The 5'-leader and 3'-trailer sequences of the pre-tRNAs are
subsequently removed by endonucleolytic cleavage catalyzed by
RNase P and RNase Z, respectively. The trinucleotides “CCA” are
then attached to the 3'-termini of tRNAs by CCA-adding enzyme.
tRNAs also undergo modification events to create many different
J Mol Biol & Mol Imaging - Volume 1 Issue 2 - 2014
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Kirino et al. © All rights are reserved
Citation: Shigematsu M, Honda S and Kirino Y. Transfer RNA as a Source of Small Functional RNA. J Mol Biol
& Mol Imaging. 2014;1(2): 8.
Yohei Kirino
Austin Publishing Group
A
B
Precursor tRNA
UU
U
Displace intiation factors
U
RNase Z
D-loop
Other function
Promote SG formation/
increase stress sensitivity
UU
U
U
3′-U tRF
eIF4G
4A
4E
3′-trailer
5′-leader
5′-leader-exon
tRF
Translational inhibition
AAAA
A
C
C
Inhibit apoptosis
Cyt c Inhibit apoptosis
Interact with ribosome
T-loop
STOP
TSEN
Mature tRNA
Dicer
A
C
C
Loaded onto AGO
3′-CCA tRF
A
C
C
A
C
C
PIWI
Xrn2
AA A A
ANG, Dicer
Promote cell proliferation
Guide RNase Z-cleavage of
target RNA
Anticodon
loop
5′-half
Stimulate Xrn2 by PIWI-binding
A
C
C
AGO
UU
5′-tRF
Promote virus replication
Intron
UU
TSEN
ANG, Dicer
3′-half
RNase Z
Promote p53 phosphorylation
S
AA A A
S
S
p53
S
S
S
Figure 1: (A) tRNA fragments derived from precursor or mature tRNAs and their processing enzymes. (B) Reported molecular functions of tRNA fragments.
non-canonical bases at various positions. Finally, the resultants
mature tRNAs are aminoacylated by their cognate aminoacyl-tRNA
synthetases, and participate in translation on the ribosome. Both pretRNAs and mature tRNAs serve as substrates for the production of
tRNA fragments.
Following the proposed nomenclature [14], tRNA fragments
identified so far can be classified into two groups: tRNA halves and
tRNA-derived fragments (tRFs)[11-15]. tRNA halves are composed
of 30–35 nt fragments derived from either the 5'- or 3'-part of fully
matured tRNAs with processed 5'- and 3'-CCA termini (Figure 1).
Shorter than tRNA halves, tRFs range from 13–20 nt in length, and
can presently be sub classified into four subgroups: 5'-tRFs, 3'-CCA
tRFs, 3'-U tRFs, and 5'-leader-exon tRFs (Figure 1). The 5'-tRFs and
3'-CCA tRFs correspond to the 5'- and 3'-parts of mature tRNAs
containing processed 5'- and 3'-CCA termini, respectively. Since
5'-leader sequences are absent, 5'-tRFs are formed by cleavage in
the D-loop after RNase P-catalyzed removal of 5'-leader sequences.
The presence of CCA sequences in 3'-CCA tRFs indicates that the
cleavage in the T-loop that produces the 3'-CCA tRFs occurs after
3'-terminal maturation by CCA-adding enzyme. Because these tRFs
and tRNA halves coexist (e.g., 5'-tRFs were purified together with 5'tRNA halves in Dicer-immunoprecipitates) [17-21], the tRNA halves,
as well as mature tRNAs, may also be a direct precursor of these
tRFs. The 3'-U tRFs are derived from the 3'-leader sequence of pretRNAs that harbor 3'-terminal uridine stretches, which result from
transcriptional termination by RNA polymerase III at thymidine
tracts. The 5'-leader-exon tRFs, another class of precursor-derived
tRFs, include the 5'-leader and the 5'-part of mature tRNAs.
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tRNA Halves as Stress-responsive Elements
Eukaryotic tRNA halves were first identified in Tetrahymena as
starvation-induced molecules [22]. Their expression was subsequently
shown to be triggered in a wide variety of organisms by a variety of
stress stimuli, such as oxidative stress, heat/cold shocks, and UV
irradiation [21, 23-30]. Therefore, tRNA halves are also known as
tRNA-derived stress-induced RNAs (tiRNAs) [25], although they
are also detected under non-stressed conditions [17, 31-33]. In
mammalian cells, Angiogenin (ANG), a member of the RNase A super
family, was discovered to be the enzyme that cleaves the anticodon
loops of mature tRNAs to produce tRNA halves [25, 26]. Rny1p, a
member of the RNase T2 family, is responsible for this anticodon
cleavage in yeast [24]. RNH1, an ANG inhibitor interacting with
ANG in the cytoplasm, was shown to be a regulatory factor for ANG
cleavage[25]. Interestingly, a tRNA modification and the enzymes
that catalyze it were also shown to be regulatory factors for the
production of tRNA halves. A number of tRNAs can be methylated
by Dnmt2 and NSun2 methyltransferases; this modification protects
the modified tRNAs from stress-induced cleavage [19, 34, 35]. It is
noteworthy that the stabilities and abundances of the two fragments
supposedly produced from the same anticodon cleavage, the 5'- and
3'-tRNA halves, can be asymmetric depending on the organism and
the conditions[19, 21, 26, 31].
Fascinatingly, stress-induced tRNA halves promote the
phosphor-eIF2α-independent formation of stress granules (SGs)
[36]. Because an SG encompasses stalled translation pre-initiation
complexes together with poly A-tailed mRNAs for translational
repression and future translational induction [37], tRNA halves may
J Mol Biol & Mol Imaging 1(2): id1007 (2014) - Page - 02
Yohei Kirino
Austin Publishing Group
Table 1: Summary of the tRNA fragment studies that investigated molecular functions and/or associated proteins of tRNA fragments.
Function
tRNA fragment used
for functional analysis
Urinary bladder
carcinoma cells
Inhibit endothelial cell growth
Mixture
[16]
Fibroblast
5′-half
ANG
Induce stress and lead to cell
death
AspGUC
[19]
U2OS
5′-half
ANG
Inhibit translation
Mixture
[25]
Organism
Material
Human
U2OS
Detected tRNA
fragment
5′-half
Fibroblast
5′-half
HeLa
5′-tRF
THP-1
3′-CCA tRF,
3′-U tRF
5′-half, 3′-half,
5′-tRF, 3′-tRF
B cell lymphoma 3′-CCA tRF
Mouse
AlaAGC, GlyCCC,
Promote SG assembly
ValAAC/CAC
Increase stress sensitivity
TyrGUA
and lead to cell death
(3′-half without 5′-P)
Inhibit translation initiation
Mixture,
by displacing eIF4G/A and eIF4F AlaAGC, CysGCA
ANG
ANG
U2OS
HEK293
Responsible Associated
nuclease
protein
ANG
Dicer,
RNase Z
AGO
AGO1, 2
5′-tRF
Dicer
3′-U tRF
RNase Z
MDA-MB-231
5′-half, 3′-half,
5′-tRF
HEK293
5′-half
MEF
5′-half
MEF
5′-half
[39]
[44]
GlnCUG
[46]
Inhibit translation
SerUGA
[51]
[52]
Inhibit translation, suppress cell
GlyGCC
proliferation by RPA1 repression
Dicer
HEK293
5′-half, 5′-tRF
AGO
[36]
Inhibit translation
AGO1, 2, 3
HCT116
A549
YB-1
Reference
Promote cell proliferation
[53]
HisGUG, LeuCAG
[55]
SerUGA
[58]
HIWI2
[59]
ANG
RNase Z
Cytc
Inhibit translation,
promote RSV replication
Cleave target RNA, modulate
apoptosis
by PPM1F repression
GluCUC
[66]
GluGUC
[70]
Inhibit translation
Mixture
[25]
Inhibit apoptosis
ArgACG
[38]
Promote p53 phosphorylation
upon oxidative stress
TyrGUA
[43]
NSC-34
5′-leader-exontRF TSEN
Testis
5′-tRF
MARWI
[60]
BmN
5′-half, 5′-tRF, 3′half, 3′-CCA tRF,
3′-U tRF
AGO2
[18]
Trypanosoma cruzi
5′-half
TcPIWI
[62]
Tetrahymena thermophila
5′-tRF
3′-CCA tRF
TWI12
Marmoset
Bombyx mori
Haloferax volcanii
Cucurbita maxima
Arabidopsis
thaliana
Phloem sap
3′-CCA tRF
TWI12
5′-tRF, 3′-tRF
Ribosome
5′-half, 3′-half
5′-tRF,
3′-CCA tRF
[64]
[47]
[45]
AGO1, 2, 4, 7
play an important signaling role in regulating gene expression by
targeting translation initiation complex. The ability to promote SG
assembly varies depending on the species of the tRNA halves. Only
5'-tRNA halves, not 3'-tRNA halves, show SG formation activity; a
5'-tRNA half from tRNAAla shows the strongest activity [36]. These
observations raise the possibility that tRNA halves might interact with
specific factors through the certain sequence motifs within tRNA.
This implies that the generation of tRNA halves may be controlled in
both qualitative and quantitative ways to produce different amounts
of selected tRNA half-species for adaptation against different stresses.
Recent studies provide compelling evidence that the enhanced
expression of tRNA halves is involved in human disease as a source
of stress response molecules. NSun2, an RNA methyltransferase
whose genetic mutations are associated with neurodevelopmental
disorders, methylates a majority of expressed tRNAs to generate the
m5C modification [19]. tRNAs lacking this modification in NSun2Submit your Manuscript | www.austinpublishinggroup.com
[63]
Localize TWI12 in nuclear
and activate Xrn2
Inhibit peptide bond formation in
ValGAC
translation
[50]
mutated patient fibroblasts or NSun2-deficient mice are more
susceptible to anticodon cleavage by ANG. As a result, 5'-tRNA
halves accumulate in NSun2-deficient cells, which is both necessary
and sufficient to trigger cellular stress responses in those cells.
Because cellular stress responses often precede apoptosis, it is not
surprising that stress responses activated by the accumulation of the
tRNA halves result in increased apoptosis in the neurons of NSun2deficient mice [19]. Contrary to their causative effect on apoptosis,
tRNA halves produced from ANG cleavage have also been reported
to inhibit apoptosis by interacting with cytochrome c in osmoticallystressed mouse embryonic fibroblasts [38].
The other example of disease-related tRNA fragments has been
observed in cells with defective CLP1, which is a multifunctional kinase
whose genetic mutations are found in neurodegenerative diseases [3941]. CLP1 associates with the tRNA-splicing endonuclease (TSEN)
complex and contributes to tRNA splicing by phosphorylating the 5'J Mol Biol & Mol Imaging 1(2): id1007 (2014) - Page - 03
Yohei Kirino
ends of 3'-tRNA halves, which are themselves the products of TSEN
complex-catalyzed removal of the anticodon intron of pre-tRNAs[42].
CLP1-deficient mice experience neurodegeneration and accumulate
a 5'-leader-exon tRF derived from tRNATyr that leads to oxidative
stress-induced cell death by promoting p53 phosphorylation [40,
43]. The catalytically-inactive CLP1 mutant derived from patients
with neurological diseases impairs TSEN-cleavage of pre-tRNA
and increases the sensitivity to oxidative stress [39, 40]. Shaffer et
al. further demonstrated the toxicity of the 5'-phosphate-lacking 3'tRNA half from tRNATyr in fibroblasts and neural cells [39]. Taken
together, these results suggest that accumulation of aberrant tRNA
fragments resulting from deficient CLP1 and TSEN activities could be
involved in the pathogenesis of neurological diseases.
Global Translational Inhibition Induced by
tRNA Halves and tRFs
When ANG has been reported to be responsible for the
production of tRNA halves under stress conditions, transfection of
5'-tRNA halves, but not 3'-tRNA halves, has been shown to inhibit
global translation in human cells [25]. Ivanov et al. then revealed
the molecular mechanism of the translational inhibition: a 5'-tRNA
half from tRNAAla displaces eIF4G/A and eIF4E/G/A (eIF4F) from
uncapped RNA and m7G-capped RNA, respectively [44]. YB-1, a
multifunctional DNA/RNA-binding protein, was found to strongly
associate with the 5'-tRNA half and mediate the translational
inhibition and SG formation. Interestingly, a terminal oligo-G
motif present in certain 5'-tRNA halves, such as the 5'-tRNA halves
from tRNAAla and tRNACys , has been shown to be required for the
translational repression [44]. Because the tRNA halves detected in the
cells are not limited to those that contain the motif, it remains to be
determined whether or not tRNA halves utilize other mechanisms for
translational regulation. Translational inhibition induced by tRNA
halves were also suggested in plant. The phloem sap of pumpkin
contains many 5'- and 3'-tRNA halves, and sap-derived RNA exhibits
global translational inhibition [45]. The translational inhibition is
likely to be mediated by tRNA halves (or nicked-tRNAs), because the
study also showed that RNase-treated, fragmented tRNAs inhibited
translation.
In addition to tRNA halves, tRFs have also been reported to be
involved in global translational inhibition in human cells. A 19 nt 5'tRF derived from tRNAGln decreased the expression of a reporter gene
that did not have a sequence complementary to that of the 5'-tRF,
suggesting that non-specific translational repression is mediated by
this 5'-tRF [46]. It is intriguing that the conserved “GG” dinucleotide
in the 3'-region of this tRF is required for translation repression.
Because 5'-tRFs were detected in the polysome fraction, interaction
of 5'-tRFs with the ribosome may contribute to translation repression
[46]. Indeed, a 5'-tRF derived from tRNAVal , one of the most abundant
tRFs in halophilic archaea, associates with the ribosome and represses
translation by interfering with peptide bond formation [47].
tRFs Bound to Argonaute Family Proteins
Although the above-mentioned translational inhibitions are not
sequence-specific and their mechanisms are completely different
from that of miRNA-mediated regulation, it has become apparent
that some tRNA fragments act as miRNAs or other small regulatory
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RNAs by binding to Argonaute family proteins. Argonaute family
proteins can be divided into two sub-clades: AGO and PIWI [2-4].
AGO proteins are ubiquitously expressed in all tissues and bind
to miRNAs and siRNAs that are 20–23 nt in length. In contrast,
PIWI proteins are predominantly expressed in the germ line (and
sometimes in cancers [48]), and interact with 26–31 nt PIWIinteracting RNAs (piRNAs). miRNAs and siRNAs are processed
from hairpin-structured or double-stranded precursor RNAs by
Dicer endonuclease, while piRNAs are believed to be generated from
long single-stranded RNAs by Dicer-independent biogenesis.
The development of antibodies against Argonaute family
proteins has enabled the Immunoprecipitation of small RNA
fractions associated with these endogenous proteins. This has
allowed identification of many tRNA fragments, as well as the abovementioned small regulatory RNAs, as Argonaute-binding small
RNAs. Immunoprecipitation of AGO proteins identified significant
amounts of tRNA fragments in Drosophila [49], Bombyx [18], and
Arabidopsis [50]. In human, 5'-tRFs, 3'-CCA tRFs, and 3'-U tRFs
have been reported to bind to AGO proteins [51-54]. Although
mature tRNAs do not meet the structural criteria for canonical
Dicer substrates, the biogenesis of some tRFs have been reported to
be dependent on Dicer in human [51, 53, 55, 56] and mouse [57].
Alternatively, ANG or other enzymes were proposed to be responsible
for Dicer-independent biogenesis of AGO-bound tRFs [54]. RNaseZ
cleavage produces 3'-U tRFs from pre-tRNAs in a Dicer-independent
manner [51, 58]. Thus, tRFs produced through both Dicer-dependent
and-independent path ways have been reported to bind to AGO
proteins. It is noteworthy that tRFs are asymmetrically loaded onto
respective AGO proteins [51, 52, 54]. For example, among the four
human AGO proteins (AGO1-4), 3'-CCA tRFs and 3'-U tRFs were
shown to preferably associate with AGO3 and AGO4 compared to the
other two [51]. This asymmetric loading of tRFs onto different AGO
proteins could have significance when considering their function and
biogenesis pathway.
Immunoprecipitation of PIWI proteins has also led to the
identification of tRNA fragments in human [59], marmoset [60],
Drosophila [61], Trypanosoma [62], and Tetrahymena [63, 64],
suggesting that tRNA fragments are widely conserved PIWI-binding
factors. The biogenesis mechanisms leading to these piRNAs-like
tRNA fragments remain to be determined. A PIWI protein essential
for growth in Tetrahymena, TWI12, binds tightly to 3'-CCA tRFs
[63]. TWI12 itself is proposed to recognize the tertiary structure of
full-length tRNAs and cleave them within the T-loop to generate
3'-CCA tRFs. The production of 3'-CCA tRFs and their interaction
with TWI12 are necessary to stabilize, localize, and stimulate Xrn2
exonuclease for nuclear RNA processing, indicating a novel role for
tRFs in RNA metabolism [64].
Targeted Gene Silencing by tRFs and tRNA
Halves
When tRFs are bound to AGO proteins, they are expected to
act as miRNAs and repress specific gene expression by recognizing
complementary target mRNAs [2-4]. Therefore, targets for these
tRFs can be explored through bioinformatics approaches or through
biochemical purification of AGO-bound target RNAs. As in the
case of miRNAs [2-4], imperfect tRF-target RNA base pairing could
J Mol Biol & Mol Imaging 1(2): id1007 (2014) - Page - 04
Yohei Kirino
induce translational silencing, whereas perfect base pairing could
trigger exonucleolytic decay of the target mRNAs.
One of the first studies describing the function of AGO-tRF
complexes was obtained from virus research. It has been long known
that retroviruses use host tRNA as a primer for reverse transcription
during the first step of retroviral replication. Yeng et al. showed that an
18 nt 3'-CCA tRF derived from one such tRNAs, tRNALys , associates
with AGO2 and targets the primer-binding site (PBS) of human
immunodeficiency virus type 1 (HIV-1) [65]. This 3'-CCA tRF directs
AGO2-mediated cleavage of the complementary PBS sequence,
thereby silencing PBS-containing reporter gene and an HIV-1 gene.
Because endogenous retroviral sequences are found extensively in the
human genome and 3'-tRFs are highly complementary to them [54],
tRF-directed pathways may also have a role in silencing endogenous
viral elements. While these reports suggest tRFs act as negative factors
for viral expression, positive effects of tRFs on viral replication have
also been reported. Infection with human respiratory syncytial virus
(RSV) leads to accumulation of 5'-tRFs that are generated by ANGmediated cleavage at sites adjacent to 5'-end of the anticodon-loop
[66]. At least one of the accumulated 5'-tRFs, a 5'-tRF derived from
tRNAGlu, has been shown to repress reporter gene expression and
promote RSV replication. As another example of a tRF with a positive
effect, a 3'-CCA tRF from tRNAPro has been suggested to function as
a primer for reverse transcription of human T-cell leukemia virus-1
(HTLV-1) [67].
Maute et al. also reported an example of a tRF that functions as
a miRNA in mature human B cells [53]. In these cells, a 22nt 3'-CCA
tRF derived from tRNAGly is generated in a Dicer-dependent manner
and associated with AGO proteins. The 3'-CCA tRF was shown to
repress the expression of target mRNAs in a sequence-specific
manner. RPA1, an essential gene for DNA dynamics and repair,
was identified as the endogenous target containing complementary
sequences in its 3'-UTR. Indeed, expression of this tRF suppresses cell
proliferation and modulates the response to DNA damage, indicating
the clear biological importance of this tRF [53]. While the endogenous
targets and biological functions of most of AGO-binding tRFs remain
obscure, Haussecker et al. used reporter assay to confirm that AGObound 3'-CCA tRFs and 3'-U tRFs silenced complementary target
RNAs [51]. One of the tRFs used in their analysis, a 3'-U tRF derived
from tRNASerUGA, was also reported to promote cell proliferation by
Lee et al.[58], which validates its cellular role, although it is unknown
whether or not interaction with AGO is required for the function of
this tRF. Accumulation of further evidence of the functionality and
endogenous biological roles of AGO-bound tRFs could result in clearcut designation of these tRFs as small regulatory RNA molecules.
In addition to the AGO-bound tRFs, tRNA fragments are
implicated in the AGO-independent sequence-specific silencing of
target RNAs by binding to tRNA processing enzymes. Nashimoto
et al. showed that RNase Z, when associated with a small guide
RNA (sgRNA), can cleave target mRNAs bearing a binding site
complementary to the sequence of sgRNAs [68, 69]. Intriguingly,
this silencing, referred to as TRUE (tRNase ZL-utilizing efficacious)
silencing, can utilize 5'-tRNA halves as sgRNAs in vitro and in vivo
for efficient silencing of complementary target RNAs [70]. PPM1F
mRNA was identified as one of the endogenous targets of a 5'-tRNA
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half derived from tRNAGln. These findings imply a physiological
role for the complex of RNase Z and 5'-tRNA halves in apoptosis,
because both RNase Z and PPM1F are implicated in the regulation
of apoptosis.
Future Perspectives
An increasing number of reports have revealed the abundant
expression of functional tRNA fragments; thus, it now appears highly
plausible that cells use tRNAs as a source for small functional RNAs to
modulate biological processes beyond translation. However, research
on tRNA fragments is still in the beginning stage; information
regarding tRNA fragment expression profiles is still fragmented,
and the molecular basis behind the biogenesis and function of tRNA
fragments remain elusive.
The immediate focus should be to capture the comprehensive
repertoire of tRNA fragments in different tissues and cells by
creating libraries and accurately profiling them or by re-evaluating
available NGS data sets. However, the biological properties of
tRNA fragments could make these attempts challenging. One such
property is the presence of post-transcriptional modifications
with in tRNA fragments. Because tRNA fragments contain noncanonical modified nucleotides of mature tRNAs, many of which
inhibit Watson-Crick base paring and thus cause arrest of reversetranscription or abnormal read through by reverse transcriptase [71],
using tRNA fragments to prepare cDNA could result in mutations or
severe reductions in the quantity of the resulting cDNAs. Therefore,
tRNA fragments from heavily modified mature tRNAs could be
underrepresented in sequencing data. This problem is inevitable with
any sequencing technology used for detection and quantification of
RNA, because there is no experimental methodology to remove all
tRNA modifications. Another property that must be considered is
the terminal modification of tRNA fragments. The tRNA fragments
derived from the 3'-part of mature tRNAs, such as 3'-tRNA halves
and 3'-CCA tRFs, contain an amino acid at their 3'-end. Furthermore,
the 5'-part of ANG-generating fragments such as 5'-tRNA halves
could contain a cyclic-phosphate at their 3'-end [72]. The preparation
of cDNA without adequate procedures to remove these terminal
modifications will result in severe under representation of these
tRNA fragments. If tRNA fragments are produced from the 5'-leader
sequences of pre-tRNAs, their 5'-end would contain a tri phosphate
modification [43]. Normal RNA sequencing methods do not
include a procedure to remove 5'-triphosphates, which may be one
of the reasons why tRFs from 5'-leader sequences have not yet been
widely discovered. It is crucial to produce libraries and interpret
sequencing data from these biochemical perspectives and to confirm
the observations using less-biased alternative techniques, such as
northern blot or tRNA microarray [28, 73].
To discriminate functional tRNA fragments from “meaningless”
degradation products, it is imperative to biochemically elucidate the
factors in their biogenesis and to determine their molecular functions.
This could be achieved by analyzing their localization, identifying the
proteins with which they interact, and examining various biological
phenomena in their presence and absence. Regarding localization,
for example, 3'-U tRFs accumulate in the cytosol, although their
biogenesis occurs in the nucleus [74], suggesting that 3'-UtRFsare
actively exported and exert their function in the cytoplasm. As
J Mol Biol & Mol Imaging 1(2): id1007 (2014) - Page - 05
Yohei Kirino
AGO-binding property of RNAs speculates miRNA-like role for the
RNAs, identification of interacting proteins is particularly important
to address the biological role of tRNA fragments. Bioinformatics
studies with support from biochemical assays have identified
numerous novel tRNA-interacting proteins [75], implying that
the biological functions of tRNAs and their fragments may be way
beyond our expectations. Further combination of computational and
biochemical efforts will significantly advance our understanding of
functional tRNA fragments and expand our knowledge regarding the
tRNA world.
Acknowledgments
We are grateful to the members of the authors’ lab for helpful
discussions. Work in the lab on this topic was supported in part by
a grant (GM106047 to YK) from the National Institutes of Health.
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Citation: Shigematsu M, Honda S and Kirino Y. Transfer RNA as a Source of Small Functional RNA. J Mol Biol
& Mol Imaging. 2014;1(2): 8.
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